The mammary gland is a complex organ which is under a myriad of regulatory controls. These include circulating hormones and growth factors, as well as local trophic factors produced by the mammary epithelial cells (MEC) or by the stroma within which the MEC are embedded. We have made the unique observation that the cytokine tumor necrosis factor-alpha (TNF-alpha) can stimulate the proliferation and morphological differentiation of rat MEC and inhibit casein production. Intriguingly, other investigators have reported that TNF-alpha inhibits the growth of malignant breast epithelial cells. Since this cytokine is produced by some of the types of cell that form the mammary stroma, we propose to determine whether TNF-alpha is a physiological regulator of growth and differentiation in normal rat MEC, to undertake preliminary studies to assess the mechanism of its action and to determine whether the activity of TNF-alpha is altered in transformed cells. Specific aims are as follows. AIM 1. To assess whether TNF-alpha is a potential paracrine or autocrine regulator of mammary epithelial cell function. Studies will be undertaken to determine if TNF-alpha mRNA and its protein product are produced in mammary epithelial and stromal cells. If TNF-alpha is found to be synthesized by one or more cell types in the mammary gland, its hormonal regulation will be investigated. AIM 2. To determine whether the effects of TNF-alpha on the proliferation and differentiation of the MEC are direct or are mediated by another cytokine. These studies will focus on how quickly MEC respond to TNF-alpha by examining the time course of its effect on early response genes such as c- myc, c-fos and c-jun, as well as on overall DNA synthesis. Additionally, potential mediation by other cytokines or growth factors will be assessed by determining whether they are synthesized in response to TNF-alpha, the time course of such a response, and if neutralizing antibodies to these cytokines block and the cytokine itself mimics, the effects of TNF-alpha on proliferation and/or differentiation. AIM 3. To determine if TNF-alpha stimulates mammary gland development in vivo. In these studies, Elvax pellets of TNF-alpha will be implanted in the mammary fat pad of immature or of ovariectomized mature rats, and development of the implanted gland compared with that of the contralateral gland. AIM 4. To determine the mechanism of the inhibitory effect of TNF-alpha on casein synthesis by examining the effect of TNF-alpha on stability of beta-casein mRNA and on the casein protein family. A determination will also be made whether the inhibitory effect of TNF-alpha is specific for casein, or whether other markers of functional differentiation (lipid synthesis, expression of WAP and transferrin) are inhibited as well. AIM 5. The effect of TNF-alpha on proliferation and differentiation of transformed rat mammary epithelial cells will be investigated both in primary culture as well as in vivo.